CN103175972B

CN103175972B - Atopic dermatitis marker and technique of using the same

Info

Publication number: CN103175972B
Application number: CN201310066108.6A
Authority: CN
Inventors: 宫田智; 宫崎香; 安田知永; 岩松明彦; 池泽善郎; 相原道子; 森山佳谷乃
Original assignee: Fancl Corp
Current assignee: Fancl Corp
Priority date: 2005-10-21
Filing date: 2006-10-19
Publication date: 2015-05-20
Anticipated expiration: 2026-10-19
Also published as: CN103175972A; CN103149368A; US20090263792A1; CN101292032A; JPWO2007046463A1; CN103149367A; WO2007046463A1; US7820396B2; HK1182770A1; HK1182768A1; JP5282228B2; HK1182769A1; KR20080063326A; CN103278641A; CN103149368B; CN103149367B

Abstract

It is an object of the present invention to find substances that can be used as disease markers for atopic dermatitis and the present invention provides a method for determining atopic dermatitis, including measurement of the expression of specific proteins and/or their genes in skin cells and/or skin tissues, wherein the specific proteins change their expression with inflammation caused by atopic dermatitis or change their expression according to the degree of predisposition to atopic dermatitis. The present invention also provides a kit for determining the degree of inflammation of atopic dermatitis or risk of developing atopic dermatitis, as well as a method for determining substances effective in the treatment and/or prevention of atopic dermatitis.

Description

Atopic dermatitis marker and utilize technology

The application is application number is 200680039191.6, the applying date is on October 19th, 2006, application people be FANCL CORPORATION, Miyazaki's perfume, Chi Ze is apt to youth, mutually former line and Sen Shanjia paddy and is, denomination of invention is the divisional application of the application of " atopic dermatitis marker and utilize technology ".

Technical field

The present invention relates to atopic dermatitis marker and utilize technology.

Background technology

Atopic dermatitis for genetic reasons, the many factors such as environmental factor and diseases induced, its mechanism is still not clear.At present because atopic dermatitis is diagnosed mainly through visual inspection, with subjective consciousness on clinical medicine, therefore lack objectivity.Particularly in atopic dermatitis, judge that the severity of the fash determining methods for the treatment of is very difficult problem.Also because society upper to be usually used in treatment of atopic dermatitis huge pillar steroids external preparation taboo trend increasingly strongly, so wish the methods for the treatment of that is applicable to and suitable dose.Therefore, explore as sb.'s illness took a turn for the worse, improve index, the performance of result for the treatment of can be very important problem by the atopic dermatitis marker of atopic dermatitis severity classification.

At present, atopic dermatitis marker uses IgE.But the disease of the antibody titer rising of display IgE, except atopic dermatitis, also has bronchial astehma, cirrhosis etc., even if so the value for antibody of IgE presents high level, also may not be just certain relevant with atopic dermatitis.There is a neodoxy recently, point out to increase because SCCA1 expresses in the skin, blood of atopic dermatitis patients, so can be used as new biomarker thing (non-patent literature 1).Also have about the very effective report of serum markers (non-patent literature 2 and 3) as atopic dermatitis patients such as NGF, Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 (Substance P), IL-16.In addition also have and use the inflammation part of atopic dermatitis and the skin of non-inflammation part, the biomarker of atopic dermatitis is carried out to the method for complete investigation.Such as, based on the gene expression analysis using DNA microarray, the disease markers of qualification atopic dermatitis connection gene has applied for patent (patent documentation 1 and 2).In addition, also have following report, cultivate obtain from the skin histology of atopic dermatitis patients epidermal keratinocytes, after fibrous bud cell, from cell, extract protein, after being separated by two dimensional electrophoresis, identify atopic dermatitis marker (non-patent literature 4 and 5) with mass spectrometer.

But, also do not use the protein in atopic dermatitis model mice skin tissue to carry out the precedent of Proteomic analysis up to now.And to the Proteomic analysis that the atopic dermatitis of human body is carried out, be from dermal harvest cell, after Initial culture, carry out Proteomic analysis, so the situation of the atopic dermatitis of human body can be reflected immediately hardly.

Patent documentation 1: Japanese Unexamined Patent Publication 2005-110602 publication

Patent documentation 2: International Publication WO01-065259 pamphlet

Non-patent literature 1:Clin.Exp.Allergy2005; 35:1327-1333

Non-patent literature 2:Br.J.Dermatol.2002Jul; 147 (1): 71-79

Non-patent literature 3:Br.J.Dermatol.2006Jun; 154 (6): 1112-1117

Non-patent literature 4:Proteomics2004,4,3446-3455

Non-patent literature 5:Proteomi cs2006,6,1362-1370

Summary of the invention

The object of this invention is to provide objectivity label as atopic dermatitis and in Treatment and diagnosis effective material.And object of the present invention is also to provide and utilizes atopic dermatitis marker, screen the prevention of atopic dermatitis or the method for the treatment of effective composition.

Present inventor is coated with haptens to atopic dermatitis model mouse (NC/Nga mouse), and people is for bringing out atopic dermatitis.The lactoferrin process simultaneously carrying out known suppression atopic dermatitis carrys out inflammation-inhibiting.Skin histology is now used to measure the protein changed because of atopic dermatitis morbidity.Analytical approach carries out Proteomic analysis for using two dimensional electrophoresis (2-DE) and mass spectrum (MS), confirms that it changes further for the protein through qualification with Western blot.

Its result, as falling ill with atopic dermatitis, it expresses the protein increased, and identifies FABP-5, Apolipoprotein A1 (Apolipoprotein A1), vimentin (Vimentin), Rho GDI etc.In addition, as with atopic dermatitis inflammation its express the protein reduced, identify Galectin-1 ,-3 ,-4 ,-7, galactose agglutinin (Galectin) class of-8 (Galectin-1 ,-3 ,-4 ,-7 ,-8) etc., desmin (Desmin), moesin (Moesin), ezrin (Ezrin), radixin (Radixin) for cytoskeletal protein, also have Annexin A2 (Annexin II), Enolase 1 (Enolase1), FABP-4, PARK7, HSP70, HSP90 etc.The great majority of these protein demonstrate contrary change when carrying out lactoferrin process again after carrying out haptens process, show that atopic dermatitis has good correlativity with or without morbidity and the increase and decrease expressed.

And, for analyzing the correlativity between atopic dermatitis marker and human body atopic dermatitis degree of inflammation found as previously mentioned, the volunteer sending out patient and health from atopic dermatitis use with it can non-invasively, safety, gather cuticular cutin tester (checker) easily and gather cuticula, therefrom extract protein, measured the expression of atopic dermatitis marker by western blot method.Its result, confirm the increase with atopic dermatitis degree of inflammation, the expression of seralbumin (Serum albumin), immunoglobulin G (Immunoglobulin G), Annexin A2 (Annexin II), Apolipoprotein A1 (Apolipoprotein A1), FABP-5, Enolase 1 (Enolase1), Galectin-7 (Galectin-7) also increases.In addition, as the protein that its expression of increase with atopic dermatitis degree of inflammation reduces, arginase I (ArginaseI), uracil dna glycosylase (Uracil-DNA glycosylase) is identified.

Main contents of the present invention are as follows.

1. judge the method for atopic dermatitis, it comprises the expression and/or its gene expression that measure following specified protein in Skin Cell and/or skin histology, and in described method, described specified protein changes with its expression of inflammation of atopic dermatitis.

Specified protein has following 23 kinds:

(1) protein selected from the Galectin group that Galectin-1 (Galectin-1), CBP-35 (Galectin-3), Galectin-4 (Galectin-4), Galectin-7 (Galectin-7), Galectin-8 (Galectin-8) form

(2) protein selected from the HSP group that HSP70, HSP90 form,

(3) protein selected from the cytoskeletal protein group that vimentin (Vimentin), Rho GDI, desmin (Desmin), moesin (Moesin), ezrin (Ezrin), radixin (Radixin) form

(4) protein selected from the FABP group that FABP-4, FABP-5 form,

(5) seralbumin (Serum albumin), immunoglobulin G (Immunogl obulin G), Annexin A2 (Annexin II), Apolipoprotein A1 (Apolipoprotein A1), Enolase 1 (Enolase1), PARK7, arginase I (ArginaseI), uracil dna glycosylase (Uracil DNA glycosylase) is also had in addition.

2.1. the method described in, wherein, Skin Cell and/or skin histology are the cuticula of the skin with the collection of cutin tester.

3.1. the method described in, it measures the expression of specified protein on protein level.

4.1. the method described in, it measures the gene expression of specified protein on rna level.

5. for judging the kit of atopic dermatitis, it comprises for the following specified protein in Skin Cell and/or skin histology, the reagent of the expression of the described specified protein that its expression of inflammation measuring adjoint atopic dermatitis changes.

Specified protein has following 23 kinds:

(2) protein selected from the HSP group that HSP70, HSP90 form,

(4) protein selected from the FABP group that FABP-4, FABP-5 form,

(5) seralbumin (Serum albumin), immunoglobulin G (Immunoglobulin G), Annexin A2 (Annexin II), Apolipoprotein A1 (Apolipoprotein A1), Enolase 1 (Enolase1), PARK7, arginase I (Arginase I), uracil dna glycosylase (Uracil DNA glycosylase) is also had in addition.

6.5. the kit described in, it possesses the cutin tester for gathering Skin Cell and/or skin histology.

7.5. the kit described in, wherein, reagent be can specific recognition with atopic dermatitis inflammation its express the antibody of specified protein changed.

8.5. the kit described in, wherein, reagent is it can express the nucleic acid probe of the mRNA specific hybrid of the specified protein changed with the inflammation of adjoint atopic dermatitis.

9.5. the kit described in, wherein, reagent for can with its nucleic acid primer of expressing the mRNA specific hybrid of the specified protein changed of inflammation of atopic dermatitis and 1 pair of nucleic acid primer that can form with the nucleic acid primer of the cDNA specific hybrid being templated synthesis with described mRNA.

10. qualification is to treatment and/or the method for preventing the effective material of atopic dermatitis, and it comprises following operation:

A operation that () makes Skin Cell and/or skin histology contact with measured matter,

B () cultivates Skin Cell contacted with measured matter in operation (a) and/or the operation of skin histology at the appointed time,

C () measures the expression of following specified protein and/or the operation of its gene expression in the Skin Cell and/or skin histology cultivated in operation (b), in described operation, described specified protein changes with its expression of inflammation of atopic dermatitis, and

D (), by comparing expression and/or its gene expression of specified protein in the expression of specified protein and/or the Skin Cell of its gene expression and contrast and/or skin histology that measure in operation (c), evaluates the operation of measured matter for the expression of specified protein and/or the effect of its gene expression in Skin Cell and/or skin histology.

Specified protein has following 23 kinds:

(2) protein selected from the HSP group that HSP70, HSP90 form,

(4) protein selected in the FABP group that FABP-4, FABP-5 form,

(5) seralbumin (Serum albumin), immunoglobulin G (Immunoglobulin G), Annexin A2 (Annexin II), Apolipoprotein A1 (Apolipoprotein A1), Enolase 1 (Enolase1), PARK7, arginase I (ArginaseI), uracil dna glycosylase (Uracil DNA glycosylase) is also had in addition.

By the present invention, the diagnosis of atopic dermatitis can be established and measure the evaluation system of its severity.Use this evaluation system, compared with previous methods, severity and the onset risk of atopic dermatitis can be judged more objectively.In addition, the present invention is also provided for judging that the severity of atopic dermatitis and the kit of onset risk and qualification have the method for the composition suppressing atopic dermatitis inflammatory effect.

Accompanying drawing explanation

Fig. 1 represents the state of the mouse skin of use 4 kinds of experiment conditions ((1) haptens (-)/lactoferrin (-), (2) haptens (-)/lactoferrin (+), (3) haptens (+)/lactoferrin (-), (4) haptens (+)/lactoferrin (+)).

Fig. 2 (a) represents the result (experiment condition of haptens (-)/lactoferrin (-) and haptens (+)/lactoferrin (-)) using the skin histology of atopic dermatitis model mouse to carry out two dimensional electrophoresis.

Fig. 2 (b) represents the result (experiment condition of haptens (-)/lactoferrin (+) and haptens (+)/lactoferrin (+)) using the skin histology of atopic dermatitis model mouse to carry out two dimensional electrophoresis.

Fig. 3 represents the expression change of galactose agglutinin in the skin histology of atopic dermatitis model mouse.

Fig. 4 represents cytoskeletal protein and the change of HSP protein expression in the skin histology of atopic dermatitis model mouse.

Fig. 5 represents protein expression change in the skin histology of atopic dermatitis model mouse.

Fig. 6 represents the result that the protein of expressing increase in the sample to atopic dermatitis morbidity uses mass spectrometer (MALDI TOF-MS) to identify.

Fig. 7 represents protein expression change in the skin histology of atopic dermatitis patients.

Fig. 8 (a) (b) represents the severity of atopic dermatitis patients and the correlationship of protein expression amount.

Result quantitative for the expression intensity of each 5 kinds of label protein in each sample to Enolase 1 (Enolase1), FABP-5, SCCA2, Apolipoprotein A1 (Apolipoprotein A1), seralbumin (Serum albumin) is concluded by the severity difference of patient by Fig. 9 (a) (b) expression.

Figure 10 (a)-(f) represents healthy person and has in the people of atopic dermatitis medical history through the correlationship between skin loss of water amount and marker representation amount.

Figure 11 (a)-(f) represents healthy person and has the correlationship in the people of atopic dermatitis medical history between horn cell area and marker representation amount.

Embodiment

Below illustrate in greater detail embodiments of the present invention.

The invention provides a kind of method judging atopic dermatitis, it comprises the expression and/or its gene expression that measure specified protein in Skin Cell and/or skin histology.Specified protein changes with its expression of inflammation of atopic dermatitis.

The mode of " expression changes " can be that the presence or absence of the expression of protein and/or its gene changes, increase and decrease etc. occurs expression.

Specified protein is preferably from Apolipoprotein A1 (Apolipoprotein A1), FABP-4, FABP-5, vimentin (Vimentin), Rho GDI, Annexin A2 (Annexin II), Enolase 1 (Enolase1), Galectin-1 (Galectin-1), CBP-35 (Galectin-3), Galectin-4 (Galectin-4), Galectin-7 (Galectin-7), Galectin-8 (Galectin-8), PARK7, desmin (Desmin), moesin (Moesin), ezrin (Ezrin), radixin (Radixin), HSP70, HSP90, seralbumin (Serum albumin), immunoglobulin G (Immunoglobulin G), select in the group that arginase I (ArginaseI) and uracil dna glycosylase (Uracil-DNA glycosylase) form.The protein selected can be a kind, also can be multiple.

The specified protein of above-mentioned 23 kinds is as follows:

(1) protein selected from galactose agglutinin (Galectin) group that Galectin-1 (Galectin-1), CBP-35 (Galectin-3), Galectin-4 (Galectin-4), Galectin-7 (Galectin-7), Galectin-8 (Galectin-8) form

(2) protein selected from the HSP group that HSP70, HSP90 form,

(4) protein selected from the FABP group that FABP-4, FABP-5 form,

The secretory protein of Apolipoprotein A1 (Apolipoprotein A1) to be molecular mass be 30,778Da, to promoting that cholesterol works from the outflow tissue or cholesterol to the reverse conveying of liver.Known its is present in the secretory protein in chylomicron, plasma high density lipoprotein level (plasma HDL) in a large number, in skin histology, and expression in chronic eczema (with the systematicness of epidermis plump and parakeratosis for feature).Gene sequence information (Apolipoprotein A1, Nucleic Acids Res.11:2827-2837 (1983), P02647).Amino acid sequence information (Apolipoprotein A1, Biochem.Biophys.Res.Commun.80:623-630 (1978), M27875).

FABP-5 (FABP-5: Fatty acidbinding protein-5) is molecular mass is 15, the intracellular protein of 033Da, the protein be mainly present in epidermis, identified because expressing rising in the tissue of chronic eczema (with the systematicness of epidermis plump and parakeratosis for feature).Be the intracellular protein be combined with fatty acid (oleic acid), hydrophobic ligands, participate in the picked-up of fatty acid, conveying, metabolism, also participate in the propagation of cell, differentiation etc.The Keratinocytic FABP-5 of breeding mesocuticle expresses few, but rises to about 2 times by induction.Combine with the S100A7 (Ca-dependent signal protein) of high expressed in chronic eczema, be positioned in talin (focaladhesion) spline structure in epidermal keratinocytes differential period.Gene sequence information (Fatty acid bindingprotin5, J.Invest.Dermatol.99:299-305 (1992), BC070303).Amino acid sequence information (Fatty acid-binding protein, epidermal, Biochem.J.302:363-371 (1994), Q01469).

The cytoskeletal protein of vimentin (Vimentin) to be molecular mass be 53,520Da is the protein of composition classIII type intermediate filament.Net distribution on a large scale in many mesenchymal cells, gives cell intensity to tackle external mechanical pressure.By its structure of phosphorylated regulation.The also known conjugated protein that there is plectin, connection silk-fibroin (synemin).Gene sequence information (Vimentin, Nucleic Acids Res.18:6692-6692 (1990), M14144).Amino acid sequence information (Vimentin, Electrophoresis18:588-598 (1997), P08670).

Rho GDI (RhoGDP-dissociation inhibitor 1) (RhoGDP-dissociation inhibitor1) is molecular mass is 23, the intracellular protein of 207Da, at epidermal keratinocytes differentiation phase high expressed, inhibition regulates Rho protein from GDP type to the conversion of GTP type.Therefore, by making Rho GDI forced expression in cell, make the disappearance such as stress fiber, talin, intracellular actin cytoskeleton system crash.Gene sequence information (RhoGDP-dissociationInhibitor1, Exp.Cell Res.209:165-174 (1993), X69550).Amino acid sequence information (Rho GDP-dissociation inhibitor, P52565).

The intracellular protein of Annexin A2 (Annexin II) to be molecular mass be 38,473Da, it is reported, its part is to cell exocrine.It is the membrane bound protein controlled by calcium, and this protein bound two calcium ions, are positioned near cell membrane.In 2 groups of annexin (annexin) iterons, 1 is combined with calcium, and 1 is combined with phosphatide.This protein or phosphatide is combined on cell membrane actin, cytoskeletal protein are cross-linked, or activate plasminogen by tPA (tissue-type plasminogen activator: tissue plasminogen activator).Gene base sequence information (Annexin A2, Gene95:243-251 (1990), BC015834).Amino acid sequence information (Annexin A2, J.Biol.Chem.266:5169-5176 (1991), P07355).

The intracellular protein of Enolase 1 (Enolase1) to be molecular mass be 47,038Da, has 2-phospho-D-glycerate=phosphoenolpyruvic acid+H ₂o (2-phospho-D-glycerate=phosphoenolpyruvate+H ₂o) enzymatic activity, works in glycolysis system.It is reported, its a part with cell proliferation, allergy relevant.In white blood cell, nerve, control the activation of the plasminogen of cell surface, participate in fibrinous formation.Magnesium is needed when there is stable dimeric structure.Although be positioned in tenuigenin, and homodimer is only had to be present in cell membrane.α-enolase (α-enolase) is expressed in most tissue, and β-enolase (β-enolase) is only expressed in musculature, and γ-enolase (γ-enolase) is only expressed in nerve fiber.Gene sequence information (Alpha enolase, Proc.Natl.Acad.Sci.USA.83:6741-6745 (1986), M14328).Amino acid sequence information (Alpha enolase, Enzyme Protein48:37-44 (1995), P06733).

The intracellular protein of Galectin-1 (Galectin-1) to be molecular mass be 14,585Da, also to cell exocrine, is present in heart, stomach, skeletal muscle, nerve, thymus gland, kidney, placenta etc.With combinations such as beta galactose glycosides, CD45, CD3, C D4.Known its affects the Effect of promoting growth of cell, the induction of cell death and immune response.In addition, be combined with the constituent of the extracellular matrix of Laminin ELISA, integrin etc., cell receptor specificities, to cell adherence, mobilely have a huge impact.Gene sequence information (Galectin-1, J.Biol.Chem.264:1310-1316 (1989), BT006775).Amino acid sequence information (Galectin-1, J.Biochem.104:1-4 (1988), P09382).

The intracellular protein of CBP-35 (Galectin-3) to be molecular mass be 35,678Da is the galactose specific agglutination element be combined with IgE, main express be found in large intestine epithelium, in activated macrophage.It is reported, CBP-35 (Galectin-3) is produced by epidermal keratinocytes, is present in the islet cell surface of skin, is combined with IgE, immunity moderation system.Gene sequence information (Galectin-3, Proc.Natl.Acad.Sci.USA.87:7324-7328 (1990), AB006780).Amino acid sequence information (Galectin-3, P17931).

The intracellular protein of Galectin-4 (Galectin-4) to be molecular mass be 35,941Da.(Galectin-4) is separated Galectin-4 Cong Jie Intestinal JEG-3 T84, is present in the adhesion part between cell and matrix, intercellular adhesion part.Gene sequence information (Galectin-4, Eur.J.Biochem.248:225-230 (1997), AB006781).Amino acid sequence information (Galectin-4, P56470).

The intracellular protein of Galectin-7 (Galectin-7) to be molecular mass be 14,944Da.In general, between cell, between cell and extracellular matrix, control the propagation of cell, be and apoptosis-related protein, the activity of control JNK, the release of cromoci.Also to tenuigenin, core, cell exocrine.Be the member of the galactose agglutinin subfamily be cloned the earliest at human epidermal, learn from the research of cultivating epidermal keratinocytes, galactose agglutinin 7 (Galectin-7) by the impact of keratinization degree, is not expressed in all epidermal cells.Gene sequence information (Galectin-7, Dev.Biol.168:259-271 (1995), L07769).Amino acid sequence information (Galectin-7, J.Biol.Chem.270:5823-5829,1995, P47929).

The intracellular protein of Galectin-8 (Galectin-8) to be molecular mass be 35,539Da.Galectin-8 (Galectin-8) is expressed in liver, heart, muscle, kidney, brain etc. much tissue, and specific expressed in prostate cancer, is not present in normal prostatic, benign prostatauxe portion.Substantially be not present in embryo, but have very high expression in each tissue of maturation.Gene sequence information (Galectin-8, Proc.Natl.Acad.Sci.U.S.A.93:7252-7257 (1996), X91790).Amino acid sequence information (Galectin-8, O00214).

The intracellular protein of PARK7 to be molecular mass be 19,891Da.PARK7 is identified as the protein relevant with Parkinson's disease, but also confirms that thereafter it participates in the re-epithelialization effect (re-epithelialization) in skin wound healing process.And from its spatial structure, also illustrate that it may work as proteinase.Gene sequence information (Parkinsondisease7, Biochem.Biophys.Res.Commun.231:509-513 (1997), BC008188).Amino acid sequence information (DJ-1protein, Q99497).

The cytoskeletal protein of desmin (Desmin) to be molecular mass be 53,405Da is in the one keeping the classIII type intermediate filament worked in eucaryotic cell structure, intensity.Be the condensate of fibrillar polypeptide, be present in smooth muscle, striated muscle.Gene sequence information (Desmin, Gene78:243-254 (1989), U59167).Amino acid sequence information (Desmin, P17661).

The intracellular protein of ezrin (Ezrin) to be molecular mass be 69,268Da.Following moesin (Moesin), radixin (Radixin) are molecules of the same clan.Main plaing a part connects cytoskeletal protein and cell membrane.Be positioned the inner side being called as the filopodia of microvillus (microvilli) of cell membrane, form the microvillus (microvilli) of enterocyte, by tyrosine kinases phosphorylate.Gene sequence information (Ezrin, J.Biol.Chem.264:16727-16732 (1989), X51521).Amino acid sequence information (Ezrin, Biochem.Biophys.Res.Commun.224:666-674 (1996), P15311).

The intracellular protein of radixin (Radixin) to be molecular mass be 68,564Da.Main plaing a part connects cytoskeletal protein and cell membrane.Gene sequence information (Radixin, Genomics16:199-206 (1993), L02320).Amino acid sequence information (Radixin, P35241).

The intracellular protein of moesin (Moesin) to be molecular mass be 67,689Da.Main plaing a part connects cytoskeletal protein and cell membrane.It is reported, express in the epidermal keratinocytes of abnormal differentiation and reduce.Gene sequence information (Moesin, Proc.Natl.Acad.Sci.USA.88:8297-8301 (1991), M69066).Amino acid sequence information (Moesin, Proc.Natl.Acad.Sci.USA.88:8297-8301 (1991), P26038).

The intracellular protein of FABP-4 (FABP-4: Fatty acid-binding protein-4) to be molecular mass be 14,588Da.Have the effect to adipocyte conveying lipid, be mainly combined with intracellular fatty acid, retinoic acid, the differential period relying on adipocyte expresses increase, has high homology with FABP-5.Gene sequence information (Fatty acid-binding protein, adipocyte, Biochemistry28:8683-8690 (1989), BT006809).Amino acid sequence information (Fatty acid-binding protein, adipocyte, P15090).

The intracellular protein of HSP70 (heat shock protein 70: Heat shock protein70) to be molecular mass be 70,052Da, works as molecular chaperones usually in cell, participates in folding, the conveying of protein, assembles and degraded etc.And the effect of HSP70 in mitochondria, ER makes protein normally shift, also work in coordination with HSP90 and participate in intracellular signal transmission.Gene base sequence information (Heat shock70kDa protein1A, Immunogenetics32:242-251 (1990), BC002453).Amino acid sequence information (Heat shock70kDa protein1, P08107).

The intracellular protein of HSP90 (heat shock protein: Heat shock protein90) to be molecular mass be 85,453Da.When cell meets with high temperature, HSP90 change structure, the molecular chaperones as the irreversible denaturation preventing other protein works.Also there is following report, in certain cell, participate in signal transmission.Gene base sequence information (Heat shock protein90, Nucleic Acids Res.17:7108-7108 (1989), X15183) amino acid sequence information (Heat shock protein90, J.Biol.Chem.264:2431-2437 (1989), P07900).

Seralbumin (Serum albumin) is the molecular mass contained in a large number in serum is the secretory protein of 69,367Da.The basic function of seralbumin (Serum albumin) is the maintenance of the colloid osmotic pressure of blood and the transport of all materials.Gene sequence information (Serum albumin, Proc.Natl.Acad.Sci.U.S.A.79:71-75 (1982)., V00494).Amino acid sequence information (Serum albumin, FEBS Lett.58:134-137 (1975)., P02768).

IgG: the secretory protein of immunoglobulin G (Immunoglobulin G) to be molecular mass be 42,632Da, accounts for 75% of the total immunoglobulin (Ig) of human body.The physiologically active of IgG has the activation of complement, the enhancing of macrophage picked-up, placenta by property etc.In addition, immunoglobulin G (Immunoglobulin G) is the large subunit (H chain) of Immunoglobulin IgG, by the classification converting expressing of gene.Exist in a large number in serum, long half time, form Multiple Antibodies molecule together with light chain (L chain), in immune response, play central role.Gene sequence information (Immunoglobulin gamma, Nucleic Acids Res.16:11824-11824 (1988)., X14356).Amino acid sequence information (Immunoglobulin gamma, Protein Sci.13:2819-2824 (2004)., P12314).

The intracellular protein of arginase I (ArginaseI) to be molecular mass be 34,735Da is the unidirectional response enzyme making L-arginine be hydrolyzed to L-Orn and urea.Be positioned in liver, in kidney, brain, mammary gland, skin, also have denier to exist.Argininemia can be caused, mental retardation, spastic quadriplegia during shortcoming.Gene sequence information (Arginase typeI, Proc.Natl.Acad.Sci.U.S.A.84:412-415 (1987)., AY074488).Amino acid sequence information (Arginase-1, P05089).

The intracellular protein of uracil dna glycosylase (Uracil-DNA glycosylase) to be molecular mass be 34,645Da.When cytosine deamination in DNA forms uracil, base-pair induced mutation can be formed with adenine.For anti-phenomenon here, the N-glycosidic bond of the BrdU that uracil dna glycosylase hydrolytic deaminzation base generates on.There are 2 kinds of hypotypes.Hypotype I, is positioned in muscle in mitochondria as Tissue distribution at intracellular targeting.Hypotype II great expression in the histocyte core of propagation.Gene sequence information (Uracil-DNA glycosylase, EMBO be (1989) J.8:3121-3125., X15653).Amino acid sequence information (Uracil-DNAglycosylase, EMBO be (1989) J.8:3121-3125., P13051).

Above-mentioned protein can be precursor protein, also can be maturation protein, can be cutting-type, also can be not cutting-type.Precursor protein can be front albumen, front former albumen.In front albumen, front former albumen etc., what have also has signal peptide.

Method of the present invention, can measure the expression of specified protein in Skin Cell and/or skin histology, also can measure its gene expression.Such as, available Northern blotting, RT-PCR method, western blot method, Immunohistochemical assay, ELISA method, antibody chip, cDNA microarray, FRET (fluorescence resonance energy transfer) (FRET; Fluorescence resonance energy transfer) mensuration such as determination method.

For measuring the expression of specified protein at protein level, specific recognition can be used as the antibody of the protein of determination object.Antibody can be any one in monoclonal antibody, polyclonal antibody.These antibody can use known method manufacture, also can be commercially available product.When measuring by western blot method, antibody can be used ¹²⁵i protein label A, peroxidase-conjugated IgG etc. carry out secondary detection.When measuring with Immunohistochemical assay, the marks such as antibody using fluorescence pigment, ferritin, enzyme.

For measuring the gene expression of specified protein at rna level, can use can with the nucleic acid probe of the mRNA specific hybrid of the protein as determination object (when measuring with Northern blotting), or also can use can with the nucleic acid primer of the mRNA specific hybrid of the protein as determination object and a pair nucleic acid primer that can form with the nucleic acid primer of the cDNA specific hybrid being templated synthesis with described mRNA (when use PCR method measures).Nucleic acid probe and nucleic acid primer can design according to the gene information of the protein as determination object.Nucleic acid probe is advisable with about 15 ~ 1500 bases usually.The marks such as nucleic acid probe using radiation element, fluorchrome, enzyme.Nucleic acid primer is advisable with about 15 ~ 30 bases usually.

In the present invention, with or without the expression of specified protein and/or its gene expression in detection of skin cell and/or skin histology, also can measure expression.With or without the expression of protein and/or its mRNA, by assigned position with or without there is spot, bar brings confirmation.The expression of protein and/or its mRNA measures by the staining power of spot, band.Or also can to protein and/or its mRNA quantitative.In order to detect most gene expression simultaneously, most protein expression, preferably can use DNA array (being fixed on substrate by probe) (NATURE REVIEWS, DRUG DISCOVERY, VOLUME1, DECEMBER2002, 951-960) protein-chip (antibody is fixed on substrate) (NATURE REVIEWS, DRUG DISCOVERY, VOLUME1, SEPTEMBER2002, 683-695), streaming dot matrix instrument (Luminex) (NATURE REVIEWS, DRUG DISCOVERY, VOLUME1, JUNE2002, detection method 447-456) etc.

Skin Cell and skin histology can from becoming the measured body judging object, and the biological species of measured body can be the mammal etc. of people, pig, monkey, gorilla, dog, ox, rabbit, rat, mouse etc.

By method of the present invention, for judging atopic dermatitis, skin biopsy sample or the Skins culture cell therefrom obtained, Skins culture tissue etc. can be used.Skin biopsy sample as described in aftermentioned embodiment, can use the sample gathered by the cuticula adhesive tape of skin (cutin tester).

Skin Cell can be enumerated as epidermal keratinocytes, skin fiber sprout cell, islet cells, melanocyte, fat cell, endothelial cell, sebaceous cell, hair papilla cell, hair matricyte etc.Skin Cell by known method from dermal harvest [(for the new cell culture experiments method of molecular biology research, p57-71, Yang Tushe, 1999) Japanese original name: " the new Fine born of the same parents of molecular biology research め train Raising experiment method; p57-71, Yang Tushe, 1999 "] ］.

Skin histology can be enumerated as the cuticula, epidermis, corium etc. of skin.Skin histology by known method from dermal harvest (Acta.Derm.Venereol., 85,389-393,2005).

Skin biopsy sample can be cell, also can be tissue.Skin biopsy sample, as described in aftermentioned embodiment, can use the keratoderma etc. that the adhesive tape of cutin tester etc. gathers.Cutin tester is used to gather cuticula sample all the time, this cuticula sample is for measuring cuticular parakeratosis degree and cell area, the degree of roughness of evaluation skin and cuticular renewal speed [(progress of the serviceability assessment technique of cosmetics and vision of the future, Japanese cosmetic operator meeting, Yao Shi newspaper office, p95-96) Japanese original name: " change make-up product useful property Evaluation value skill Intraoperative and enter Walk と prospect in the future; Japanization make-up product skill Intraoperative person meeting; Yao Shi Reported society, p95-96) " ］].As can in consulting (counseling) shop, gather cuticula sample simply, safely in oneself, non-invasively to obtain method of protein from cuticula very effective.

As one of example of the present invention, atopic dermatitis can use following standard determination.

As use the various antibody of the Enolase 1 (Enolase1) shown in embodiment 1 (Fig. 8), FABP-5, SCCA2, Apolipoprotein A1 (Apolipoprotein A1), albumin (Albunin) and Annexin A2 (Annexin II) etc. analysis example shown in, skin samples is obtained with it from the volunteer of atopic dermatitis morbidity, measure the expression of multiple label protein expression amount or its gene, disclose the relation of symptom and expression.

From atopic dermatitis send out the expression of the same area (non-patient's control sample) of the site of pathological change (analysis sample) of patient and non-site of pathological change (sending out patient's control sample) and atopic dermatitis non-patient relatively, degree, the pathogenic factor of decision analysis sample atopic dermatitis symptom.So, label protein analysis result can to present multiple symptom atopic dermatitis diagnosis, treatment work.

The present invention is also provided for the kit judging atopic dermatitis.Kit of the present invention comprises for the specified protein in Skin Cell and/or skin histology, the reagent that the described specified protein that its expression of inflammation measuring adjoint atopic dermatitis changes is expressed.

Such as, comprise in kit of the present invention can specific recognition with atopic dermatitis inflammation its express the reagent of the antibody of specified protein changed.

Specified protein is preferably from Apolipoprotein A1 (Apolipoprotein A1), FABP-4, FABP-5, vimentin (Vimentin), Rho GDI, Annexin A2 (Annexin II), Enolase 1 (Enolase1), Galectin-1 (Galectin-1), CBP-35 (Galectin-3), Galectin-4 (Galectin-4), Galectin-7 (Galectin-7), Galectin-8 (Galectin-8), PARK7, desmin (Desmin), moesin (Moesin), ezrin (Ezrin), radixin (Radixin), HSP70, HSP90, seralbumin (Serum albumin), immunoglobulin G (Immunoglobulin G), select in the group that arginase I (ArginaseI) and uracil dna glycosylase (Uracil DNA glycosylase) form.

The a complete set of reagent, operation instructions etc. of the protein on the adhesive tape, immuno-chemical method detection adhesive tape of dermal tissue harvesting also can be comprised in kit.In operation instructions except recording the using method of kit, also can record the criterion etc. of atopic dermatitis.

Also have an example in addition, such as, kit of the present invention comprise as reagent can with atopic dermatitis inflammation its express the nucleic acid probe of the mRNA specific hybrid of the specified protein changed.

Specified protein is preferably from Apolipoprotein A1 (Apolipoprotein A1), FABP-4, FABP-5, vimentin (Vimentin), Rho GDI, Annexin A2 (Annexin II), Enolase 1 (Enolase1), Galectin-1 (Galectin-1), CBP-35 (Galectin-3), Galectin-4 (Galectin-4), Galectin-7 (Galectin-7), Galectin-8 (Galectin-8), PARK7, desmin (Desmin), moesin (Moesin), ezrin (Ezrin), radixin (Radixin), HSP70, HSP90, seralbumin (Serum albumin), immunoglobulin G (Immunoglobulin G), select in the group that arginase I (ArginaseI) and uracil dna glycosylase (UracilDNA glycosylase) form.

Also can comprise in kit for the adhesive tape of dermal tissue harvesting, for extracting the reagent class of RNA, the reagent class, operation instructions etc. with Northern Northern blot analysis RNA from the skin histology on adhesive tape.In operation instructions, except recording the using method of kit, also record the criterion etc. of atopic dermatitis.

Also have an example, kit of the present invention comprise as reagent can with its nucleic acid primer of expressing the mRNA specific hybrid of the specified protein changed of inflammation of atopic dermatitis and a pair nucleic acid primer that can form with the nucleic acid primer of the cDNA specific hybrid being templated synthesis with described mRNA.

Specified protein is preferably from Apolipoprotein A1 (Apolipoprotein A1), FABP-4, FABP-5, vimentin (Vimentin), Rho GDI, Annexin A2 (Annexin II), Enolase 1 (Enolase 1), Galectin-1 (Galectin-1), CBP-35 (Galectin-3), Galectin-4 (Galectin-4), Galectin-7 (Galectin-7), Galectin-8 (Galectin-8), PARK7, desmin (Desmin), moesin (Moesin), ezrin (Ezrin), radixin (Radixin), HSP70, HSP90, seralbumin (Serum albumin), immunoglobulin G (Immunoglobulin G), select in the group that arginase I (ArginaseI) and uracil dna glycosylase (Uracil DNA glycosylase) form.

Also can comprise in kit for the adhesive tape of dermal tissue harvesting, for extracting the reagent class of RNA from the skin histology on adhesive tape, analyzing the reagent class, operation instructions etc. of RNA by RT-PCR method.In operation instructions, except recording the using method of kit, also record the criterion etc. of atopic dermatitis.

The present invention also provides qualification to have the method for the material for the treatment of and/or the effect of prevention atopic dermatitis, and the method comprises following operation:

C () measures the expression of specified protein and/or the operation of its gene expression in the Skin Cell and/or skin histology cultivated in operation (b), in described operation, described specified protein changes with its expression of inflammation of atopic dermatitis, and

Measured matter can be any material, can be enumerated as protein, peptide, vitamin, hormone, polysaccharide, compound sugar, monose, low molecular compound, nucleic acid (DNA, RNA, oligonucleotides, mononucleotide etc.), lipid, native compound than that described above, synthesis compound, plant extracts, the separator of plant extracts, the potpourri etc. of above-mentioned substance.

Skin Cell and skin histology described above.

The contact of measured matter and Skin Cell and/or skin histology, any method can be used, such as, the upper method etc. of cultivating Skin Cell and/or skin histology of culture vessel or culture plate (sheet) can be the method for being added to by measured matter in the nutrient solution of Skin Cell and/or skin histology, being coated with or being fixed with measured matter.Also can be the biosome of the mammal (such as mouse, rat, cavy, rabbit, pig etc.) beyond end user or people etc., measured matter directly be coated the method on skin, oral administration to the method for measured matter.

The incubation time of Skin Cell and/or skin histology is not particularly limited, as long as can confirm that measured matter has the unfruitful time to the expression of specified protein in Skin Cell and/or skin histology and/or its gene expression.

Such as, during Skin Cell use human body normal epidermal keratinocytes, be advisable with 12 ~ 48 hours, be preferably 12 ~ 24 hours.Herein, " cultivate Skin Cell and/or skin histology " instigates Skin Cell and/or skin histology to grow the meaning of propagation, wherein, except make isolated after Skin Cell and/or skin histology grow except propagation, also comprise and make to have the biosome etc. that Skin Cell, the biosome its own existence of skin histology, raising and cultivation have Skin Cell, skin histology.

As the Skin Cell and/or the skin histology that compare contrast, can be the Skin Cell before contacting with measured matter and/or skin histology, also can be except not contacting with measured matter, carry out Skin Cell and/or the skin histology of same treatment.

As an example of the present invention, in the Skin Cell contacted with measured matter and/or skin histology, Apolipoprotein A1 (Apolipoprotein A1), FABP-5, vimentin (Vimentin), Rho GDI, Annexin A2 (Annexin II), Enolase 1 (Enolase1), in seralbumin (Serum albumin) or immunoglobulin G (Immunoglobulin G), the expression of any one reduces compared with the control, there is measured matter can be evaluated as while the effect that any one protein expression above-mentioned is reduced, it is the material with treatment and/or the effect of prevention atopic dermatitis that this measured matter can be accredited as.

In addition, an other example of the present invention, in the Skin Cell contacted with measured matter and/or skin histology, FABP-4, Galectin-1 (Galectin-1), CBP-35 (Galectin-3), Galectin-4 (Galectin-4), Galectin-7 (Galectin-7), Galectin-8 (Galectin-8), PARK7, desmin (Desmin), moesin (Moesin), ezrin (Ezrin), radixin (Radixin), HSP70, HSP90, the expression of arginase I (ArginaseI) or uracil dna glycosylase (Uracil DNA glycosylase) increases compared with the control, there is measured matter can be evaluated as while the effect that any one protein expression above-mentioned is increased, it is the material with treatment and/or the effect of prevention atopic dermatitis that this measured matter can be accredited as.

Embodiment

Below, describe the present invention in detail according to embodiment, but the invention is not restricted to following embodiment.

Embodiment 1

Material and experimental technique

1. the haptens process of atopic dermatitis model mouse (NC/Nga mouse)

Atopic dermatitis model mouse (NC/Nga mouse), buy male mice [the NC/Nga slc in 6 week age, Sankyo Labo Service Corporation (Japanese original name “ tri-Association ラボサー PVC ス Co., Ltd. ")], raise under normal conditions.NC/Nga mouse has multiple system, the system that this research uses is that when only raising under conventional environment, atopic dermatitis is not fallen ill, by the system just making atopic dermatitis fall ill after the immune inducing agent process such as DNFB (dinitrofluorobenzene: Dinitorofluorobenzene).Therefore, by the DNFB solution (haptens) of 0.1% by within 4 weeks, coating on two ears and left and right skin of back altogether for 1 time weekly.

Meanwhile, the lactoferrin that can relax atopic dermatitis inflammation is dissolved in drinking water with 1 μ g/ml to be thrown to mouse.

The experiment condition of mouse is (1) haptens (-)/lactoferrin (-), (2) haptens (-)/lactoferrin (+), (3) haptens (+)/lactoferrin (-), (4) haptens (+)/lactoferrin (+), each use 3.

2. the extraction of protein in atopic dermatitis model mice skin tissue

Cut the skin histology with haptens/lactoferrin process mouse of 4 weeks, shredded by tissue cutter, immigration determines in advance in the centrifuge tube of tare weight and measures tissue weight.Add the damping fluid of tissue weight × 0.85mg urea (urea), tissue weight × 0.1 μ l1.5%SDS, tissue weight × 0.1 μ l8.5% triton x-100 (Triton X-100), tissue weight × 0.05 μ l2ME, with HG30 homogenizer (homogenizer) (HITACHI company) homogeneous.15,000rpm (15,000xg), 10 DEG C centrifugal 30 minutes, reclaim after supernatant, then by its supernatant 50,000rpm (100,000xg), 10 DEG C centrifugal 1 hour, using its supernatant as sample.Quantitative by the amount of dot blot to protein.

3. the extraction of protein in atopic dermatitis patients skin histology

Cutin tester [AsahiBiomed Co. is pasted at the position be not inflamed near the position that atopic dermatitis patients is inflamed (mainly wrist) and inflammation part, Ltd (Japanese original name " アサヒバイオメッ De society ")], obtain cuticula.In contrast, from recovery sample the person that atopic dermatitis is not fallen ill.Cutin tester, for a position, uses 3 to reclaim in same area.Every 1 diaphragm, in the 1 × sample buffer (samplebuffer) [83mMTris-HCl (pH6.8), 2.7%SDS, 28% glycerine (glycerin)] of 50 μ l, is scraped with cell and is scraped sample thief from diaphragm.With 15000rpm (15,000xg), 4 DEG C, 10 minutes centrifugal rear recovery supernatants, DC protein determination (DC protein assay) (BIO-RAD company) is used to carry out quantification of protein.

4. two-dimensional gel electrophoresis (2-DE)

4-1 first ties up isoelectric point electrophoresis

The protein of 60g is mixed with gel inflation fluid [5M urea (urea), 2M thio urea (thiourea), 0.5% ampholyte (ampholytes) (pH3.5 ~ 10) (Amersham Biosciences company), 0.0025% orange G (Orange G), 2.5mM TBP, 1% triton x-100 (Triton X-100)], make total amount be after 340 μ l, expand a night in Immobiline immobilized ph gradient strip (Immobiline Dry-Strip gel) (18cm, pH3-10, NL) (Amersham Biosciences company) 20 DEG C.Use the device of Anatech company, the program of the first Wesy 20 DEG C, 500V-2 hour, 700V-1 hour, 1000V-1 hour, 1500V-1 hour, 2000V-1 hour, 3000V-1 hour, 3500V-10 hour carries out isoelectric point electrophoresis.After electrophoresis, gel is immersed in SDS level pad [5.8M urea (urea), 0.06M thio urea (thiourea), 0.5%dithiothereitol (DTT) (w/v), 25% glycerine (glycerol), 0.0025%BPB], equilibrate at room temperature 1 hour.

4-2 second ties up SDS-PAGE

Second dimension uses the damping fluid [Cathode buffer: 0.05M Tris, 0.05M tri-s' (methylol) methylglycine (tricine), 0.05%SDS, anode buffer liquid: 1M Tris-HCl (pH8.8)] of Tris-tri-(methylol) methylglycine (tricine) system, carries out SDS-PAGE by 18cm × 18cm, 7.5% acrylamide gel.

4-3 electroblotting method

The gel terminated after SDS-PAGE is used half dry type transfer device [Nihon Eido Co., Ltd. (Japanese original name " Japanese エイドー society ")], at the steady current transferase 12 hour of pvdf membrane [ProBlott Membranes (Applied Biosystemus company)] the upper 150mA of 20cm × 20cm.Transfering buffering liquid uses anode 1 liquid: 0.3M Tris-HCl (pH10.4), 20% methyl alcohol (methanol), anode 2 liquid: 25mM Tris-HCl (pH10.4), 20% methyl alcohol (methanol), catholyte: 25mM Tris-HCl (pH10.4), 20% methyl alcohol (methanol), 40mM6-aminocaproic acid (6-amino hexanoic acid) (pharmaceutical worker industry pure with light).With after TTBS damping fluid [20mMTris-HCl (pH7.5) (BIO-RAD company), 500mM NaCl, 0.3% Tween-20 (Tween-20) (BIO-RAD company)] washing in 20 minutes 3 times after transfer, with washing in MQW2 minute 3 times.Then film is sealed the colloidal gold solution [Colloidal Gold Total Protein Stain (BIO-RAD company)] of the full 50ml of rear leaching, vibrate and make protein staining after 1 ~ 2 hour.After film dyeing, remove colloidal gold solution, wash 1 minute, after washing 5 times with pure water, make it dry.

5. the qualification of protein

On 5-1PVDF film, the reduction S-alkylation of protein and proteinase enzyme are cut

Cut the protein spots transferred on pvdf membrane, put into test tube, add the reduction damping fluid [8M guanidine hydrochloride (guanidine-HCl) (pH8.5)] of 100 ~ 300 μ l, 0.5M Trisbase, 0.3%EDTA-2Na (w/v), 5% acetonitrile].In reduction damping fluid, add the DTT (dithiothreitol) being equivalent to 1mg of dissolving, in vitro will be replaced into nitrogen, room temperature leaves standstill 1 hour, goes back crude protein.Before reaction terminates, be added in the iodoacetic acid (Monoiodoacetic Acid) being equivalent to 3mg dissolved in 1M NaCl, under shading status, Keep agitation carries out S-carboxymethylation in 15 ~ 20 minutes.Take out pvdf membrane after end, with pure water agitator treating after 5 minutes, in 2% acetonitrile, same method stirs.Take out pvdf membrane, after moving to the in vitro cleaning 2 ~ 3 times that Lys-C enzyme cutting buffering liquid [70% acetonitrile/20mMTris-HCl (pH9.0)] is housed, immerse Lys-C enzyme cutting buffering liquid completely, enzyme cuts 1 hour.

5-2 quality analysis and peptide mapping fingerprinting

The solution dilution 7 cut by proteinase enzyme doubly makes acetonitrile concentration be 10%.As pre-treatment, in order to activate the filler part of ZipTipc18 suction pipette head (MILLIPORE company), 50% acetonitrile/0.1%TFA (trifluoroacetic acid: trifluoro-acetic acid) is repeatedly used repeatedly, with 2% acetonitrile/0.1%TFA (trifluoroacetic acid: trifluoro-acetic acid) repeatedly to suck, discharge.Then with activated Zip Tipc18 suction pipette head, proteinase enzyme is cut liquid repeatedly to suck, discharge, the peptide after fragmentation is adsorbed on pack portion.Again 2% acetonitrile/0.1%TFA (trifluoroacetic acid: trifluoro-acetic acid) is repeatedly sucked, discharges, remove salt.Suck saturated matrix solution 0.5 ~ 1 μ l dissolved in 50% acetonitrile/0.1%TFA (trifluoroacetic acid: trifluoro-acetic acid), place after about 10 seconds, be added dropwise in the attached target probe of mass spectrometer.Make sample solid, measure with mass spectrometer (MALDI-TOF MS).Based on the mass value obtained, identify with reference to the protein logged at database (MS-Fit, Mascot Search).

6.Western blotting

Use the sample being used for western blot method, carry out SDS-PAGE by the Tris-Glycin system of Laemmli.After carrying out SDS-PAGE, gel is used every 1cm ²the steady current of 0.8mA is transferred to pvdf membrane (MILIPORE company), then uses 5% skimmed milk/PBS (-) 4 DEG C to close and spends the night.After pvdf membrane 0.1% polysorbas20 (Tween20)/PBS (-) washing 3 times, at room temperature react 1 hour with 1 antibody.Secondly, the vibration 1 of 2 antibody biotinylation anti-sheep IgG (Biotinylated anti-goatIgG) (Vector Laboratories company) [1: 1000 dilution (dilution)] is constantly little, 1 hour is reacted with the avidin D (Alkaline phosphatase-conjugated avidin D) (Vector Laboratories company) (dilute at 1: 1000) of alkaline phosphatase conjugation, with the chloro-3-indolyl phosphate (5-bromo-4-chloro-3-indolylphosphate) of the bromo-4-of 0.6mg/ml5-, 1.2mg/ml nitro blue tetrazolium (nitrobluetetrazolium), 0.1M Tris-HCl (pH9.5), 5mM MgCl ₂colour developing.And 2 antibody use horseradish peroxidase-labeled anti-mouse IgG (HRP labeled anti-mouse IgG) (Amersham Bioscience company) (1: 1000 dilution), horseradish peroxidase-labeled anti-rabbit IgG (HRP labeled anti-rabbitIgG) (Amersham Bioscience company) (1: 1000 dilution), horseradish peroxidase-labeled Chinese People's Anti-Japanese Military and Political College mouse IgG (HRP labeled anti-rat IgG) (Amersham Bioscience company) (1: 1000 dilution), during the anti-sheep IgG of horseradish peroxidase-labeled (HRP labeled anti-goatIgG) (Santa Cruz company) (1: 1000 dilution), use enhanced chemiluminescence (Enhanced chemi luminescence) (ECL) (Amersham Bioscience company) kit, detected by fluorescence developing.Kind and the dilution factor of 1 antibody using are as follows: the anti-HSP70 monoclonal antibody of mouse (mouse anti-HSP70monoclonal antibody) (Santa Cruz Biotechnology company) (1: 1000 dilution), the anti-annexin I I of rabbit polyclonal antibody (rabbit anti-annexin II polyclonal antibody) (Santa Cruz Biotechnology company) (1: 1000 dilution), the anti-HSP90 monoclonal antibody of mouse (mouse anti-HSP90monoclonal antibody) (Santa Cruz Biotechnology company) (1: 1000 dilution), the anti-GRP94 monoclonal antibody of rat (rat anti-GRP94monoc lonal antibody) (Stres sgeen company) (1: 1000 dilution), the anti-Galectin-1 monoclonal antibody of mouse (mouse anti-galectin-1monoclonal antibody) (R & D Systems company) (1: 1000 dilution), the anti-CBP-35 monoclonal antibody of rat (rat anti-galectin-3monoclonal antibody) (R & D Systems company) (1: 1000 dilution), the anti-Galectin-4 monoclonal antibody of mouse (mouse anti-galectin-4monoclonal antibody) (R & D Systems company) (1: 1000 dilution), the anti-Galectin-7 monoclonal antibody of mouse (mouse anti-galectin-7monoclonal antibody) (R & D Systems company) (1: 1000 dilution), the anti-Galectin-8 monoclonal antibody of mouse (mouse anti-galectin-8monoclonal antibody) (R & D Systems company) (1: 1000 dilution), goat-anti-galactose agglutinin-9 polyclonal antibody (goat anti-galectin-9polyclonal antibody) (Santa Cruz Biotechnology company) (1: 1000 dilution), the anti-desmin polyclonal antibody of rabbit (rabbit anti-desmin polyclonal antibody) (Santa Cruz Biotechnology company) (1: 1000 dilution),Goat-anti-vimentin polyclonal antibody (goat anti-vimentin polyclonal antibody) (Santa Cruz Biotechnology company) (1: 1000 dilution), the anti-ENO1 polyclonal antibody of rabbit (rabbit anti-enolase-1polyclonal antibody) (Santa Cruz Biotechnology company) (1: 1000 dilution), anti-ezrin/radixin/moesin the polyclonal antibody of rabbit (rabbit anti-ezrin/radixin/moesin polyclonal antibody) (Chemicon company) (1: 1000 dilution), goat-anti-FABP-4 polyclonal antibody (goat anti-FABP-4polyclonal antibody) (G-T research company) (1: 1000 dilution), the anti-Apolipoprotein A1 monoclonal antibody of mouse (mouse anti-apolipoprotein A1monoclonalantibody) (ICN biomedicals company) (1: 1000 dilution), goat-anti-Apolipoprotein A1 (goat anti-apolipoprotein A1) (Abcom company) (1: 1000 dilution), goat-anti-PARK7 polyclonal antibody (goat anti-PARK7polyclonalantibody) (Abcom company) (1: 1000 dilution), the anti-Rho GDI of mouse monoclonal antibody (mouse anti-Rho GDI monoclonal antibody) (Santa Cruz Biotechnology company) (1: 1000 dilution), goat-anti-FABP-5 polyclonal antibody (goat anti-FABP-5polyclonal antibody) (R & D Systems company) (1: 1000 dilution), the anti-SCCA2 monoclonal antibody of mouse (mouse anti-SCCA2monoclonal antibody) (Santa Cruz Biotechnology company) (1: 1000 dilution), the anti-FABP-5 polyclonal antibody of rabbit (rabbit anti-FABP-5polyclonal antibody) (BioVendor company) (1: 5000 dilution),Rabbit Anti-Human blood albumin polyclonal antibody (rabbitanti-human albumin polyclonal antibody) (Inter-Cell company) (1: 1000 dilution).

7. the mensuration of horn cell area minimizing degree

By cutin tester by being pressed in disease sites, skin without stratum corneum cell.After acquiring cutin tester 1% BG of corneocyte, 0.5% gentian violet aqueous solution dyeing, use the method for digit microscope (VHX-100, KEYENCE Co, Ltd., Japan) (Gai Bian Kashihara Fuchi etc., JSCCJ, 23,1,1989.Japanese original text: Kashihara Fuchi ら method The changes, JSCCJ, 23,1,1989.) observe.The horn cell area of the image obtained divides 5 ranks (1 point: little, 2 points: slightly little, 3 points: general, 4 points: slightly large, 5 points: large) scoring by the visual assessment of professional judgement person.

8. through the mensuration of skin loss of water amount

Commercially available skin water loss tester (Tewameter) (TEWAMETER TM210, Courage+Khazaka electronic GmbH Inc.) is used to measure.Its measuring principle is that the moisture that supposition is scattered and disappeared to air from skin surface spreads according to Fick rule, measures the vapor pressure of 2 of number mm on skin, calculates the amount of moisture from epidermis evaporation.

9. by the label protein measuring of ELISA (enzyme-linked immunosorbent assay: enzyme-linked immunosorbent assay)

In 96-well elisa plate, will add with 50 μ l/well with the cuticula protein solution that phosphate buffer solution (PBS) is diluted to 0.2 μ l/ μ l, 4 DEG C of absorption 18 hours.After destratum corneum protein solution, immersion lock solution in the PBS} containing 1% bovine serum albumin(BSA) (BSA), 37 DEG C, closed 1 hour.With cleansing solution { after the PBS} washing of polyoxyethylene (20) sorbitan mono-laurate (medicine pure with light) containing 0.05%, 1 antibody-solutions { is made the various antibody of 5mg/ml } add with 50 μ l/well respectively with cleansing solution, 37 DEG C, reaction 2 hours.After washing, 2 antibody-solutions { are changed anti-mouse immunoglobulin G (VECTOR LABORATORIES) with the HRP (horseradish peroxidase: Horseradi shperoxidase) that 1mg/ml made by cleansing solution or changed anti-rabbit immunoglobulin G (VECTOR LABORATORIES) with the HRP (horseradish peroxidase: Horseradi sh peroxidase) that 1mg/ml made by cleansing solution } and adds with 50 μ l/well respectively, 37 DEG C, reaction 1 hour.After washing, enhanced chemiluminescence (Enhanced chemi luminescence) (ECL) (Amersham Bioscience company) kit is used to carry out chemiluminescence reaction, by chemiluminescence detector (SpectraMax Lmax II 384, Molecular devices) detect, quantize.

Experimental result

1. the haptens process of atopic dermatitis model mouse

In this research use atopic dermatitis model mouse (NC/Nga mouse) under conventional environment through haptens

When raising after process, haptens process, after 4 weeks, with the naked eye can confirm the dermatitis morbidity exactly liking atopic dermatitis.The lactoferrin (molecular weight that with saliva, blood in contained ferric ion be combined be the protein of 8 ten thousand) effective to atopic dermatitis is mixed in potable water and makes it drink by this research.The result of raising after haptens and lactoferrin process, the inflammation exactly liking human body atopic dermatitis is found that there is at the back of the mouse of haptens coating process, and after haptens coating process, the mouse of having drunk the water being mixed into lactoferrin further with only carry out haptens and be coated with compared with the mouse that processes, inflammation receives suppression (Fig. 1).By haptens process, falling ill with atopic dermatitis, it expresses the protein that changes as by lactoferrin process, and atopic dermatitis symptom is relaxed and returns to and level equal during non-processor, then can think that its protein is label likely.

2. use the skin histology of atopic dermatitis model mouse to carry out two dimensional electrophoresis analysis

For the qualification protein relevant with atopic dermatitis, the skin two dimensional electrophoresis of haptens coating process or untreated, lactoferrin process or untreated mouse is analyzed the protein changed.

Take out the skin (having the position of inflammation when haptens is coated with) of the further mouse of city milk ferritin after non-processor (contrast) mouse, the mouse of haptens coating mouse, in contrast city milk ferritin and coating haptens, sample buffer is added according to weight, after the fragmentation of Polytron type homogenizer, 15000rpm (15,000xg), 30 minutes centrifugal, then reclaim supernatant.By its supernatant 100,000xg, 1 hour ultracentrifugation, supernatant is used as sample.Two dimensional electrophoresis, adhesive tape (strip gel), second Wesy 7.5% acrylamide gel of its first Wesy pH3-10 carry out.Use colloidal gold solution dyes, the protein of change detected, to finding out that the protein of change uses mass spectrometer (MALDI-TOF MS) to carry out Molecular Identification (Fig. 2 (a) (b)).Its result, identifies 43 protein in 49 protein analyzed.In the protein identified, express the identification of proteins increased in the mouse (producing the mouse of atopic dermatitis inflammation) of haptens process and go out FABP-5, Apolipoprotein A1 (Apolipoprotein A1), vimentin (Vimentin) etc., agnoprotein matter has 4 kinds.On the other hand, in the mouse of haptens process, express the identification of proteins reduced and go out CBP-35 (Galectin-3), desmin (Desmin), PARK7 etc.And the great majority of the protein identified are the skelemins of keratin 5 (kerat in5), Keratin 16 (keratin16), desmin (Desmin), vimentin (Vimentin), moesin (Moesin) etc.

3. the antibody of the protein using atopic dermatitis model mouse to change confirms

For confirming the above-mentioned shown protein expression changed because atopic dermatitis morbidity, using various antibody, being analyzed by western blot method.First, containing CBP-35 (Galectin-3) in the protein of the result qualification of reason two dimensional electrophoresis, confirm so be conceived to galactose agglutinin family.Galactose agglutinin is the one of glycoprotein, does not have common burst, but by the stimulation of immune response etc. or not irriate and discharging to extracellular.Report is had to point out, each galactose agglutinin molecule shows different Tissue distribution, participate in immunological regulation, adhere between cellular matrix, cell-cell adhesion, all different physiological phenomenons such as wound healing are (with reference to J.Biol.Chem.264:1310-1316 (1989), J.Biochem.104:1-4 (1988), Proc.Natl Acad.Sci.USA.87:7324-7328 (1990), Eur.J.Biochem.248:225-230 (1997), Dev.Biol.168:259-271 (1995), J.Biol.Chem.270:5823-5829 (1995), Proc.Natl.Acad.Sci.U.S.A.93:7252-7257 (1996)).

Therefore, except CBP-35 (Galectin-3), also use comprise in galactose agglutinin (Galectin) family Galectin-1 ,-3 ,-4 ,-7 ,-8, the antibody of-9 (Galectin-1 ,-3 ,-4 ,-7 ,-8 ,-9), analyze the change (Fig. 3) of expression in atopic dermatitis model mouse skin.Its result, for Galectin-1 ,-3 ,-4 ,-7 ,-8 (Galectin-1 ,-3 ,-4 ,-7 ,-8), mouse expression compared with untreated mouse (control mice) of haptens process reduces.And on the other hand, for Galectin-1 ,-4 ,-7 ,-8 (Galectin-1 ,-4 ,-7 ,-8), even the mouse of also known haptens process, after making its city milk ferritin, the expression of mouse still returns to the level close to the untreated mouse of haptens (control mice).Galactose agglutinin-9(Galectin-9) do not change because of haptens process, so think that the correlativity of itself and atopic dermatitis is low.

Same analysis (Fig. 4) has also been carried out for cytoskeletal protein desmin (Desmin), moesin (Moesin)/ezrin (Ezrin)/radixin (Radixin), vimentin (Vimentin).The mouse of haptens process is compared with untreated control mice, the expression of desmin (Desmin) reduces, and the expression of moesin (Moesin)/ezrin (Ezrin)/radixin (Radixin) (50KDa cutting-type) and vimentin (Vimentin) increases.Particularly moesin (Moesin)/ezrin (Ezrin)/radixin (Radixin) is by haptens process, and its low-molecular-weight cutting-type obviously increases, by also not reducing after lactoferrin administration.And moesin (Moes in)/ezrin (Ezrin)/radixin (Radixin) belongs to the protein of same family, is analyzed by two dimensional electrophoresis, is accredited as moesin (Moesin).Because this antibody used identifies this antibody of 3 kinds, so think due to inflammation, moesin (Moesin) or other protein is independent or most high expressed.

For heat shock protein HSP70, HSP90, GRP94, the mouse of haptens process is compared with untreated mouse (control mice), the expression of HSP70, HSP90 reduces, but the expression of GRP94 and haptens process, non-processor have nothing to do, not change (Fig. 4).

And, for FABP-4 (FABP-4: fatty acid binding protein-4), FABP-5 (FABP-5: fatty acid binding protein-5), Enolase 1 (Enolase1), PARK7 (DJ-1), Annexin A2 (Annexin II), Apolipoprotein A1 (Apolipoprotein A1), Rho GDI, undertaken analyzing (Fig. 5) by western blot method.Its result, for Annexin A2 (Annexin II), Enolase 1 (Enolase1), FABP-4, PARK7, mouse expression compared with untreated mice (control mice) of haptens process reduces.And except FABP-4, even the mouse of haptens process, after making its city milk ferritin, the expression of mouse has still returned to the level close to the untreated mouse of haptens (control mice).On the other hand, for Rho GDI, FABP-5, Apolipoprotein A1 (Apolipoprotein A1), the mouse of haptens process is expressed and increases (Fig. 5) compared with untreated mouse (control mice).Known Rho GDI weakens intercellular adhesion, so, because atopic dermatitis this protein expression increase of falling ill may depart from relevant with the skin in its participation atopic dermatitis.

4. use the skin histology of atopic dermatitis patients to carry out SDS-PAGE analysis

From the above results, candidate's atopic dermatitis marker of Select to use atopic dermatitis model mouse, but the label need investigating that can these protein become human body atopic dermatitis.Therefore, actual same area (Control) use sending out the volunteer that the inflammation part (Fig. 6, A.P.) of patient and non-inflammation part (Control), atopic dermatitis are not fallen ill from atopic dermatitis is called the membrane-like article collected specimens of cutin tester.Reclaim the sample of 3 diaphragms from the same area of human body skin, then use 1 × SDS sample buffer to dissolve skin histology.Thereafter, the sample using atopic dermatitis to send out patient (2 people) and non-patient (3 people) carries out SDS-PAGE, and the protein of expressing increase in the sample that use mass spectrometer (MALDITOF-MS) is fallen ill to atopic dermatitis is identified.Its result shows, Annexin A2 (Annexin II), SCCA 1 (squamous cell carcinoma antigen-1) (SCCA1), squamous cell carcinoma antigen 2(squamous cell carcinoma antigen-2) (SCCA2), FABP-5 (fatty acid binding protein-5 (FABP-5)), seralbumin (Serum albumin) and immunoglobulin G (Immunoglobulin G) expression increase (Fig. 6) with atopic dermatitis morbidity.On the other hand, also show that the expression of arginase I (ArginaseI) and uracil dna glycosylase (Uracil-DNA glycosylase) is sent out in patient in atopic dermatitis and reduce (Fig. 6).

5. by using the various antibody of atopic dermatitis patients skin histology to analyze

When using atopic dermatitis model mouse to pass through the analysis of western blot method, find that there is 17 kinds of protein and there occurs change.Measure these protein and whether have same change (Fig. 7) when the inflammation morbidity of human body atopic dermatitis.Simultaneously by using the SDS-PAGE of the skin histology of atopic dermatitis patients to analyze, the protein changed is inquired into.

For Annexin A2 (Annexin II), the site of pathological change (Fig. 7, A.P.) that atopic dermatitis sends out patient is expressed and is increased compared with non-site of pathological change (Control), and molecular weight is smaller compared with the band of atopic dermatitis non-patient.This reason is still not clear, but thinks that this molecular level difference may to send out the physique of patient and Fei Fa patient different relevant from atopic dermatitis.

For Enolase 1 (Enolase1), squamous cell carcinoma antigen 2(squamous cell carcinoma antigen2) (SCCA2), the site of pathological change sending out patient 1 atopic dermatitis increases.

For PARK7, because atopic dermatitis sends out the different increase and decrease that it is expressed of patient also not necessarily, but compared with atopic dermatitis non-patient, atopic dermatitis is sent out in patient and is still all shown minimizing trend.

In addition, for Apolipoprotein A1 (Apolipoprotein A1), the non-site of pathological change that atopic dermatitis non-patient and atopic dermatitis send out patient is not expressed completely, and only expresses increase at atopic dermatitis site of pathological change.

Above result is concluded in Table 1.

Table 1

6. by using the various antibody of patient skin tissue to analyze

For confirming the practicality of the atopic dermatitis marker candidate detected from the skin histology of atopic dermatitis model mouse and minority atopic dermatitis patients, use the skin samples of atopic dermatitis patients volunteer 17 (male sex 7, women 10) and the non-volunteer 15 (male sex 10, women 5) that falls ill accepting dermatologist's diagnosis and treatment, measure the correlativity of itself and atopic dermatitis severity.According to the severity of atopic dermatitis patients, (association of Japanese dept. of dermatology treatment of atopic dermatitis policy 2004 changes version, ［ Japanese original name: " association of day Ben Pi Skin section アトピー skin Skin inflammation is controlled Treatment ガイ De ライ Application 2004 and changed Order version " ］) classification, severity is 1 of 1, severity is 7 of 2, severity is 6 of 3, and severity is 3 of 4.From atopic dermatitis volunteer 17, collect the data of eosinophil leucocyte, IgE, LDH etc. in the known blood relevant to the severity of atopic dermatitis.Skin samples cutin tester gathers, and analyzes 6 kinds of protein expressions (Fig. 8 (a) (b)) by western blot method.

For Enolase 1 (Enolase1), fail to detect Enolase 1 from the skin of atopic dermatitis non-patient, but find that there is great expression in the skin of atopic dermatitis patients.In patients, compared with non-inflammation part, inflammation part is expressed in many patient 17 people 12 people.On the contrary, the patient of minimizing has 2 people.

For FABP-5 (Fatty acidbinding protein-5) (FABP-5), in non-patient's skin, substantially all fail to detect except 1 people, and at the inflammation part of atopic dermatitis patients, all find that there is the strongly expressed corresponding to severity.Compared with non-inflammation part, express in many patient 17 people at inflammation part and have 13 people.

For squamous cell carcinoma antigen 2(Squamous cell carcinoma antigen2) (SCCA2), in non-patient, 1 people is weak expression, but clearly in atopic dermatitis patients skin, expresses increase.Compared with non-inflammation part, express in many patient 17 people at inflammation part and have 9 people, on the contrary, the patient of minimizing is 1 people.Compared with FABP-5, can find out that expression there occurs deviation.

For Apolipoprotein A1 (apolipoprotein A1), in non-patient's skin, 1 people is weak expression, in the specific patient of atopic dermatitis patients, is tested with the trend of strongly expressed.Compared with non-inflammation portion, express in many patient 17 people in inflammation portion and have 9 people, the patient of minimizing is 1 people.

Seralbumin (Serum albumin), is all tested with stronger expression in all atopic dermatitis patients, and the difference between whole inflammation portions and non-inflammation portion is very little.In non-patient, except 1, express all very weak.

For Annexin A2 (Annexin II), although express weak, in the skin of atopic dermatitis patients, be tested with great expression, in non-patient, substantially do not detect expression.Compared with non-inflammation portion, express in many patient 17 people in inflammation portion and have 6 people, the patient of minimizing also has 6 people.

Except the Annexin A2 (Annexin II) that expression intensity is low, quantitative to the expression intensity of each sample of each 5 kinds of label protein of Enolase 1 (Enolase1), FABP-5, SCCA2, Apolipoprotein A1 (Apolipoprotein A1), seralbumin (Serum albumin).The severity of its result by patient is concluded, as shown in Fig. 9 (a) (b).

Because of FABP-5, seralbumin (Serum albumin), Enolase 1 (Enolase1) shows has high correlativity with patient skin atopic dermatitis severity, so can be used for the severity judging patient's atopic dermatitis.Even if seralbumin (Serum albumin) also can be detected from non-inflammation part, so it can reflect the physique of the relevant patient that to fall ill with atopic dermatitis.There is deviation in Apolipoprotein A1 (Apolipoprotein A1) and SCCA2, and Annexin A2 (Annexin II) has and expresses high feature at non-inflammation part in expression.The expression of these labels also may reflect the speciality of patient.

Above result is summarized in table 2.

Table 2

7. the checking of label validity in the people having atopic dermatitis medical history

The people that You Changqu hospital is experienced because of atopic dermatitis is about 6,340,000 people, and the people presenting the allergic skin of atopic dermatitis symptom about can be speculated as 1,200 ten thousand people.Concerning atopic dermatitis, not by means of only pharmaceutical treatment, by NMF skin care also very effectively (on field, eight is bright, Fragrance Journal, p13-19, in June, 2003), also there are plenty of such people to carry out the people of skin care with Medicines and Health Product, cosmetics.By thinking above, the purposes of label found in this research, not only can be used for dermatologist in the diagnosis of atopic dermatitis, also can be used on the skin diagnosis in consulting shop, skin diagnosis in oneself.Therefore, once there to be atopic dermatitis to fall ill, to have and accepted the artificial object having atopic dermatitis medical history that dermatologist treats experience, confirmed the practicality of atopic dermatitis marker candidate.

Characteristic as atopic dermatitis changes, very clearly there are minimizing and the existence of karyocyte in cutin (the Tagami H.et al. of the horn cell area that is hyperfunction through skin loss of water amount (TEWL), that entirely do not cause because of keratinization caused because stratum corneum barrier function declines, J.Invest.Dermatol.Symp.Proc., 6,1,87 ~ 94,2001).Therefore, the index being atopic dermatitis degree of inflammation with the minimizing degree of the hyperfunction degree of TEWL and horn cell area, detects the validity of candidate markers.

With healthy person 11, the volunteer of the people 10 of atopic dermatitis medical history is had to evaluate for object.Healthy person and have atopic dermatitis medical history people normal portions use Medial upper arm portion, (7, the elbow inside portion, position having the inflammation part of the people of atopic dermatitis medical history to use to be inflamed, 1, neck, finger 1, seam portion, 1, portion inside the knee), measure the hyperfunction degree of TEWL and the minimizing degree of horn cell area and 6 kinds of candidate markers [FABP-5, Galectin-7 (Galectin-7), Enolase 1 (Enolase1), SCCA2, Apolipoprotein A1 (Apolipoprotein A1), Annexin A2 (Annexin II)] expressivity between correlativity.The minimizing degree of horn cell area is by skin without stratum corneum decoration method, and the hyperfunction degree of TEWL is measured by skin water loss analyzer (Tewameter).The expressivity of 6 kinds of candidate markers, healthy person and have atopic dermatitis medical history people normal portions use Medial upper arm portion, there is the inflammation portion of the people of atopic dermatitis medical history to use the position be inflamed, gather cuticula by cutin tester, therefrom extract protein and measured by ELISA method.

Measure the result of the correlativity between the hyperfunction degree of TEWL and the expressivity of 6 kinds of candidate markers [FABP-5, Galectin-7 (Galectin-7), Enolase1, SCCA2, Apolipoprotein A1 (Apolipoprotein A1), Annexin A2 (Annexin II)] as shown in Figure 10.The TEWL that the X-axis of Figure 10 represents is at healthy person and have the normal portions of the people of atopic dermatitis medical history substantially equal, but shows hyperfunction at the inflammation part of the people having atopic dermatitis medical history.The expression of 3 kinds of labels of the FABP-5 that the Y-axis of Figure 10 (a) ~ (c) represents, Galectin-7 (Galectin-7), Enolase 1 (Enolase1), with healthy person and have atopic dermatitis medical history people normal portions compared with, hyperfunction hyperfunction in correlativity having the inflammation part of the people of atopic dermatitis medical history and a TEWL.On the other hand, the expressivity of 3 kinds of labels of the SCCA2 that the Y-axis of Figure 10 (d) ~ (f) represents, Apolipoprotein A1 (Apolipoprotein A1), Annexin A2 (Annexin II), for healthy person < has the normal portions < of the people of atopic dermatitis medical history to have the inflammation part of the people of atopic dermatitis medical history, very effective to the diagnosis of the risk factor of atopic dermatitis morbidity.

Measure the result of the correlativity between the minimizing degree of horn cell area and the expressivity of 6 kinds of candidate markers [FABP-5, Galectin-7 (Galectin-7), Enolase 1 (Enolase1), SCCA2, Apolipoprotein A1 (Apolipoprotein A1), Annexin A2 (Annexin II)] as shown in figure 11.The horn cell area that the X-axis of Figure 11 represents, at healthy person with there is the normal portions of the people of atopic dermatitis medical history substantially equal, but reduces at the inflammation part of the people having atopic dermatitis medical history.The expression of 3 kinds of labels of the FABP-5 that the Y-axis of Figure 11 (a) ~ (c) represents, Galectin-7 (Galectin-7), Enolase 1 (Enolase1), relevant to the minimizing of horn cell area, with healthy person and have atopic dermatitis medical history people normal portions compared with, have the inflammation part of the people of atopic dermatitis medical history show hyperfunction.On the other hand, the expressivity of 3 kinds of labels of the SCCA2 that the Y-axis of Figure 11 (d) ~ (f) represents, Apolipoprotein A1 (Apolipoprotein A1), Annexin A2 (Annexin II), for healthy person < has the normal portions < of the people of atopic dermatitis medical history to have the inflammation part of the people of atopic dermatitis medical history, very effective to the diagnosis of the risk factor of atopic dermatitis morbidity.

The present invention have found the protein of protein and the minimizing increased with the degree of inflammation of atopic dermatitis and its expression of risk factor of atopic dermatitis morbidity.By detect these protein expressions change, can according to the reason of atopic dermatitis, the relation of symptom, carry out more accurate diagnosis and the judgement to initiation potential degree.And these labels can be used for the exploitation of the medicine of atopic dermatitis, sensitive skin applies some make up and the exploitation etc. of healthy food.

Claims

1. specific recognition can prepare with its antibody of expressing the specified protein changed of inflammation of atopic dermatitis the application diagnosed or detect in the preparation of atopic dermatitis, described specified protein comprises Galectin-7.